Note: Descriptions are shown in the official language in which they were submitted.
;'7
ANTIBACTERIAL COMPOUNDS
.
This invention relates to antibacterial compounds
and in particular to a novel antibacterial compound
produced by the bacterium Pseudomonas fluorescens,
together with salts and esters of the compound.
British Patent No. 1,395,907 describes and claims :
a process for the isolation of antibacterial compounds
rom the bacterium Pseudomonas fluorescens, one such
compound being called pseudomonic acid o formula (I)
OH
~ C02~CHz)8C02
~:~ OH
which will be referred -to herein as "pseudomonic acid
A",
It has now been found that~a further antibacterial
compound:can be isolated from Pseudomonas fluorescens
and this:~compound can also be prepared from pseudomonlc
ac:id A.
15 : ~ ~Acc:ordingly the~present invention provides a
compound:of formula (II) or a pharmaceutically accept- -
able salt or ester thereof:
. .
OH
CH3 HO ~ ~ ~ ~ CO2(CH2)8c02
CH~ ~ CH3 (II)
OH
The compound (II) will be ref~erred to herein as
"pseudomonic acid C". It is believed that the compound
has the absolute steriochemistry as shown in formula
(II`I) OH
CH3 HO ~ ~
CH l l l C2(CH2)8C2H
.~` ~ CH3
~ ~III)
OH
both double bonds being in the trans - or E conigura
tion.
Suitable non-toxic sal-ts of pseudomonic acid C
include metal salts, e.g. aluminium, alkali metal
salts such as sodium or potassium, alkaline earth
metal salts such as calcium or magnesium, and ammonium
or substitu-ted ammonium salts for example those with
lower alkylamino such as triéthylamine, hydroxy-lower
alkylamines such as 2-hydroxyethylamine, bis-(2-hydrox-
ethyl)-amine or tri-(2-hydroxyethyl)-amine, cyclo-
alkylamines such as bicyclohexylamine, or with procaine,
dibenzylamine, N,N-dibenzylethylene-diamine, l-ephen-
amine, N-ethylpiperidine, N-benzyl-~ phenethyl-amine,
dehydroabietylamine, N,N'-bis-dehydroabietylethylene~
diamine, or bases of the pyridine type such as
pyridine, collidine or quinoline. ~ -
Preferred salts are alkali metal salts. Suit-
able esters incluùe: ;
~'''' ~ . :
,
~, .
(a~ Cl_20 alkyl, C2 8 alkenyl or C2 8 alkynyl each of
which may be optionally substituted by C3_7 cycloalkyl,
halogen, carboxy, Cl 6 alkoxycarbonyl, carbamoyl, aryl,
heterocyclylg hydroxy, Cl 16 alkanoyloxy, amino, mono-
s and di (Cl_6) alkylam
(b) C3 ~ cycloalkyl optionally substituted with
Cl 6 alkyl;
(c) aryl;
(d) heterocyclyl.
The term laryl' includes phenyl, and naphthyl
optionally substituted with up to five halogen, Cl 6
alkyl, Cl 6 alkoxy, halo(Cl 6)alkyl 7 hydroxy, amino,
carboxy, Cl 6 alkoxycarbonyl, or Cl 6 alkoxycarbonyl(C
(Cl_6)alkyl groups~
The term ~heterocyclyl' includes single or fused
rings comprising up to four hetero atoms in the ring
selected from oxygen, nitrogen sulphur and optiona:Lly
substituted with up to three halogen, Cl 6 alkyl,
Cl 6 alkoxy, halo(Cl 6)alkyl, hydroxy, amino, carboxy,
20 Cl 6 alkoxycarbonyl, Cl_6alkoxy-carbonyl(C1 6)alkyl,
aryl or oxo groups.
One class of ester groups comprise allcyl, aryl,
and aralkyl groups, any of which may be substituted
with a hydroxy, amino or halogen group. For e~ample
25 the ester group may be a Cl 6 alkyl group in particular,
methyl, ethyl, n or iso propyl, n, sec-, iso or tert-
butyl; a halo-(Cl 6)-alkyl group such as tri~luoro-
methyl, 2,2,2-trichloroethyl; an aminoalkyl group such
as aminomethyl, 2-aminoethyl; hydroxymethyl, hyclroxy-
30 ethyl; phenyl; substituted phenyl, or a benzyl group.
Preferred esters are Cl 6 alkyl esters.
Pseudomonic acid C, its salts and esters have
antibacterial activity. They have particularly high
activity against Haemphilus in~luenzae, _ isserla
35 gonorrhoeae and Mycoplosma sp, and are therefore of
value in the treatment o~ respiratory and venereal
diseases, and of mycoplasma-induced human ancl
-.~ . ~- .
.: . - . . ,
. ' : . .
: . .
. . . :. -. . ~,:
veterinary diseases. Furthermore, the compounds of
this invention have -the advan-tage over pseudomonic acid
A of being stable to acidic conditions, and more
stable to alkaline conditions.
In humans the infections which pseudomonic acid C
its salts and esters may be particularly useful agains-t
include venereal disease. Because it is not a ~-lactam
antibiotic it is effective against ~-lactamase-produc-
ing strains of N. gonorrhoeae, against which standard
treatments such as penicillin and cephalosporin anti-
biotics would not be useful. Pseudomonic acid C may
also be effective in the treatment of respiratory
infections such as chronic bronchitis, and bacterial
meningitis; non-specific urethritis and pneumonia. In
animals it may be employed generally as a growth
promoter, or for the ~reatment of mastitis in cattle
and for treatment of mycoplasma infections in animc~ls
such as turkeys, chickens and ~igs.
This invention also provides a pharmaceu-tical or
veterinary composition which comprises pseudomonic acid
C, or a salt or ester thereof together with a pharma
ceutically or veterinary acceptable caxrier or excipi-
ent.
The compositions may be formulated for administra
tion by any route, and would depend on the disease
being treated. The compositions may be in the form of
table-ts~ capsules, powders~ granules~ lozenges, or
liquid preparations, such as oral or sterile parenteral `
solutions or suspensions.
Tablets and capsules for oral adrninistration may
be in unit dose presentation form, and may contain
conventional excipients such as binding agents, for
example syrup, acacia, gelatin, sorbitol, tragacanth,
or polyvinyl-pyrollidone; fillers, for example lactose,
sugar, maize-starch, calcium phosphate, sorbitol or
glycine; tabletting lubricants, for example magnesium
stearate, talc, polyethylene glycol or silica;
,
, , , - :
-, . , . , . , . .
-- 5 --
disintegrants, for example po~ato starch; or acceptable
wetting agents such as sodium lauryl sulphate. The
tablets may be coated according to methods well known
in normal pharmaceutical prac-tice. Oxal liquid prep-
arations may be in the form OI ~ Ior example, aqueous
or oily suspensions, solutions, emulsions, syrups, or
elixirs, or may be presented as a clry product for
reconstitution with water or other suitable vehicle
before use. Such liquid preparations may contain con-
10 ventional additives such as suspending agents, for
example sorbitol, syrup, methyl cellulose, ylucose
syrup, gelatin hydrogenated edible fats, emulsifying
agents, for examplè lecithin, sorbitan monooleate, or
acacia; non a~ueous vehicles (which may include edible
15 oils), for example almond oil 9 fracti.onated coconut oil,
oily esters such as glycerine, propylene glycol, or
ethyl alcohol; preservatives, for example methyl or
propyl ~-hydroxybenzoate or sor'bic acid, and if desired
convention flavouring or colouring agents.
Suppositories will contain conventional suppos-
itory bases, e.g. cocoa-butter or other glyceride.
~or parenteral administration, fluid unit dosage
forms are prepared utilizing'the compound and a sterile
vehicle, water being preferred. The compound~ depend-
25 ing on t'he vehicle and concentration used, can be
either suspended or dissolved in the vehicle. In pre-
parlng solutions the compouncl can be dissolved in
water for injection and filter sterilized before fill-
ing into a suitable vial or ampoule and sealing.
30 Advantageously, adjuvants such as a local anesthetic,
p.reservative and buffering agents can be dissolved in
the vehicle. To enhance the stability the composition
can be frozen after filling into the vial and water
removed under vacuum. The dry lypophilized powder is
35 then sealed in the vial. Parenteral suspensions are
prepared in substantially the same manner except that
the compound is suspended in the vehicle instead of
, : . ............ :. .
: . - . .:
':,: . . ~. ,: , ,... .~ .. ' . . '
being dissolved and sterilization cannot be accomplished
by filtration. The compound can be s-terilized by
exposure to ethylene oxide before suspending in the
sterile vehicle. Advantageously, a surfactant or
wetting agent is included in the composition to
facilitate uniform distribution of the compound.
The compositions may contain from 0.1% to 99% by
weight, preferably from 10-60% by ~eight, of the active
material, depending on the method of administration.
Where the compositions comprise dosage units, each unit
will preferably contain from 50-5~0 mg., of the active
ingredient. The dosage as employed for adult human
treatment will preferably range from 100 mg to 3 g.,
per day, for instance 250 mg-2 g., per day, depending
on the route and frequency of adminis-tration.
Alternatively pseudomonic acid C or a sal-t or
ester may be adrninistered as part of the total dietary
intake. In this case the amount of compound employed
may be less than 1% by weight of the diet and in pre-
ferably no more than 0.5% by weight. The diet for ani-
mals may consist of normal foodstuffs to which the com-
pound may be added or it may be added to a premix.
The present invention also provides a process for
the preparation of pseudomonic acid C or a salt or ester
thereof which process comprises reacting pseudomonic
acid A or a salt or ester thereof with a r~agent which
converts an epoxide to an olefin; and optionally
carrying out one of the following steps:
(ij forming a sal-t of the pseudomonic acid C produced;
(ii~ esterifying the pseudomonic acid C or salt thereof
to produce an ester of pseudomonic acid C; or
(iii) hydrolysing an ester of pseudomonic ~cid C.
A number of reagents for converting an epoxide to
an olefin are known in the literature, and the part-
icular reagent of choice for the process of the pre-
sent invention is a matter of trial and error. Some
.. : . . . .
'. , ' . ' :
: . ~
-
such reagents are more suitable than others for this
purpose. Some generally applicable methods are as
follows:
(a) Potassium selenocyanate in methanol/water;
(see JCS Chem. CommO, 19/5, 1~16; J~S 1949~ 278j
~b) Lower valent tungsten halides; for example WC16/
butyl lithium (see J~Amer.Chem.SocO 1972,94,6538)
(c) Ph3P = Se/-trifluroacetic acid;
(see JCS Chem. Comm. 1973, 253)
~O (d) Trifluoroacetyl iodide/sodium iodide;
(see J.Org.Chem., 1978~43,1841).
Other methods are described in the followiny references:
J. ~mer. Chem. Soc., 1973, 95, 2697.
Tet. Letts (17) 1976, 1395.
Ber. 1955, 88~ 165~.
J, Org. Chem., 1958t 22, 1118.
It has been ~ouncl that one convenient method is
the use of potassium selenocyanate.
Suitable so]vents for use with potassium selen-
ocyanate include mixtures of water with alkanols, in
particular Cl-C20 alkanols. It has been found that
higher yields of the compound of formula (II) are
achieved if an alcohol is employed with a large, in
particular branched or cyclic, alkyl group. Specific
alcohols include iso-hexyl alcohol, tert-amyl alcohol
and cyclohexyl alcohol. The reaction is generally
performed at elevated temperatures, suitably at about
the boiling point o the solvent employed. The time
for which the reaction is performed depends on the temp~-
erature of the reaction, and therefore on the solvent.
Generally a time of from 2-9 days is suitable.
Another suitable method for converting the epoxide
of pseudomonic acid A, or a salt or ester thereof into
an olefin, comprises treatment with trifluoroacetyl
iodide and sodium iodide. ~he trifluoroacetyl iodide
may be prepared in situ from trifluoroacetic anhydride.
The reaction is suitably conducted at ambient
,;'
. . . . . . .
. . . :. .
. . . ' , ' ' ' . - . :
. .
temperature for ~rom about 10 to 36 hours, suitably
about 24 hours.
When the free acid or salt of pseudomonic acid
C is required it may be convenient to employ an ester
of pseudomonic acid A for the above process9 which
ester is a carboxyl-protecting group. Suitable carb-
oxyl-protecting groups would depend on the reaction
condi-tions for de epoxidation and include the 2,2,2-
trichloro-ethyl ester, (which may be removed with zinc
in a lower alcohol, espcially methanol) phenyl, penta-
chlorophenyl, benzyl, and t-butyl ester groups. Other
suitable carboxyl-protecting are silyl groups. In
this case the carboxylic acid is reac-ted with a silyl-
ating agent such as a halosilane or a silazane. A
preferred silylating agent is N,O-bis(trimethylsilyl)
acetamide, which produces the trimethyl-silyl deriva-
tive of the acid.
Prior to the above process of this invention, it
may be desirable to protect the hydroxyl groups in
pseudomonic acid A or its salt or ester. Although the
reaction is possible without hydroxyl protection, in
some cases higher yields of the pseudomonic acid C
derivative could be formed if the hydroxyl groups were
protected. Such protecting groups must be removable
under suitably mild conditions and suitable groups
include silyl groups produced from a silylating agent
as discussed above. Particularly suitable hydroxyl-
protectlng groups include tri-methylsilyl, t-butyl-
dimethylsilyl 7 methylthiomethyl. A preferred hydroxyl-
protecting group is trimethylsilyl, as it is readily
removed on completion o~ the reaction. Alternatively,
for some de-epoxidation reactions it is possible to
protect the hydroxyl groups with other ester radicals
which may be removed by chemical or enzymatic means.
Examples include p-nitrobenzoate, methoxyaceta-te,
phenoxyacetate, trifluoroacetate, each of which may be
.
.. ~ , ~ . . ........................ ,... :: ::
-
removed under mild basic conditions such as aqueous
ammonia; or potassium carbonate in aqueous methanol.
It is also possible to protect the glycol moiety
in pseudomonic acid A, and suitable reagents for form-
lng such a hydroxyl-protecting group include compounds
of formula (IV).
oR2 .,
Rl C oR3 (IV)
1 4
OR
wherein Rl is hydrogen or a Cl 6 alkyl group and R ,
R and R independently represent a Cl 6 alkyl group.
The group Rl may be for example hydrogen, methyl,
ethyl, n- or iso-propyl. Most suitably, Rl represents
hydrogen so tha-t the compound of formula (I~) is a
trialkyl orthoformate. -
Groups R2, R3, and R4 may be for example, methyl,
ethyl, n- or iso-propyl, n-, _ -, sec- or tert-
butyl. Preferably R2, R3, and R are all the same and
each represents a methyl group.
Other glycol pro-tecting groups include those
wherein the glycol moiety is converted to the structure:
o \ / ~a
o / \Rb ' '
where Ra and Rb are hydrogen, Cl 6 alkyl, or phenyl.
Preferably R and R are both methyl, i.e. the group
is the isopropylidene group. This group may be intro-
duced onto pseudomonic acid A or its salt or ester by
reaction with 2,2-dimethoxypropane, and removed by
.
.: . :
.
~ .
' ' ' ' ': ' ' ': ' . . ~ ,
-- 10 --
treatment with acetic acid.
The hydro~y-protecting group may be removed by a
conventional method for the particular hydroxyl-protect~
ing group.
It may be such that it can be removed directly or
alternatively, it may be converted into a different pro-
tecting group which is then removable under ~lifferent
conditions. This latter approach may be employed when
a glycol protecting group derived from a compound (IV)
is used; it is converted by acid to the group -OCOR
which is then removed.
When an ester of pseudomonic acid C is required,
the esterification step, step (ii) above may be per~
formed by any conventional method, for exa~ple by
reaction of the acid, or a salt thereof:-
(a) with the appropriate halide, sulphate or alkane-
sulphonate of the alcohol in the presence of a solvent
such as acetone, dimethylsulphide or dimethvlsulphoxicle
and calcium, or potassium carbonate or with the halide
in the presence of hexamethyl phosphoramide; or
(b) by phase transfer catalysis methods with the
halide and/or sulphate of the alcohol in aqueous and/or
organic solution in the presence of a quaternary ammon-
ium salt such as tetrabutyl ammonium bisulphate or
halide, or benxyltrime-thyl-ammonium halide; or
(c) with a diazoalkane.
The hydrolysis of an ester of pseudomonic acid C
(step ~iii) above) may be chemical hydrolysis, for
example by selective alkaline hydrolysis; or enzymic
hydrolysis, for example by the use of Bakersl Yeast.
Also included within the scope of the present
invention is a process for the preparation of pseudo-
monic acid C or a salt or ester thereof which comprises
yrowing Pseudomo_as fluorescens under aerobic conditions
on or in a culture medium containing inoxganic salts and
sources of asimilable carbon and nitxogen until the
culture medium exhibits at least detectable antibacterial
.: . :: : . - .
.. . , . . : . .. .:
- . . .
.
.
v`~
activity, acidifying the culture medium; extracting
with an organic solvent for the active materials
dissolved in the culture medium9 and thereafter either:
(a) separating pseudomonic acid C or a salt thereof
from any other active materials and optionally there-
after esterifying the separated acid; or
(b) esterifying the active materials, separating an
ester of pseudomonic acid C from esters of any other
active materials and optionally thereafter hydrolysing
the separated ester to form pseudomonic acid C or a
salt thereof.
In the above process, the cultivation step where
Pseudomonas fluorescens is grown is conventional.
Any strain of this organism may to our knowledge be
employed; one suitable public strain being Pseudomonas
fluorescens NCIB 10586. (NCIB = National Collection
of Industrial Bacteria).
After the fermentation is completed, the active
rnater:ials, lncludlng pseudomonic acid C, are extracted
from the acidified aqueous culture ~ledium into a suit-
able solvent.
Preferably, the extraction procedure comprises
extraction of the culture medium, after acidification,
with an organic solvent; re-extraction of the organic
extract with aqueous alkaline buffer solution; and
finally re-extraction of the latter, after acidifica-
tion, with organic solvent. Suitable solvents can
be found by trial and error; examples include iso-
butylmethyl ketone (IBMK), chloroform and preferably
ethyl acetate.
The pseudomonic acid C may then be separated
from other active materials produced in the ferment-
ation, either directly (step (a) above) or by
esterifying the mixture and separating the ester of
pseudomonic acid C (step (b) above). When Pseudo- -
monas flu~orescens NCIB 10586 is employed as the bact-
erium, the majox material which is produced in
. .
. : ;, . ..
. '
,~
- 12 -
addition to pseudomonic acid C is pseudomonic acid A.
If this latter material is present in substantial
quantities it is preferable to remove the majority
by crystallisation of pseudomonic acid A9 optionally
with seeding, from a suitable solvent for example
diethyl ether.
If alternative (a) is carried out pseudomonic
acid C may be separated directly by chromatography
from the remaining mixture, either as the free acid
itself or as a slat thereof. On chxomatography on
silica gel, pseudomonic acid C is eluted slightly
before pseudomonic acid A and the fractions can be
identified accordingly.
The separated pseudomonic acid C or a salt thereof
lS may be esterified by any of the methods described
earlier in this specification.
If alternative (b) above is carried out the
mixture of active materials is first esterified,
preferably after removing the majority of the pseudo-
monic acid A by crystallisation. Again any of the
above described methods of esterification may be
employed. It is convenient to form Cl 6 alkyl esters
of the components in the mixture, preferably me-thyl
esters.
The resultant mixture of esters may then be
subjected to chromatography and the desired ester of
pseudomonic acid C thereby separated. If the free
acid or salt is required they may be produced by
chemical or enzymatic hydrolysis of the separated
ester.
The invention is illustrated in the following
Examples.
' .
' :
. - . ,
r~ r~
-- 13 --
Example 1
Methyl 10,11-D~oxypseudomonate_A (methyl pseudomonate _)
from meth~ seudomonate A
A solution of methyl pseudomonate A (1.03 g ;
2 m.mole) and potassium selenocyanate (0.846 g ;
6 m.mole) in methanol-water 9 : 1 (30 ml) was heated
under reflux for 7 days. The black precipitate of
selenium was filtered off and the flltrate evaporated
- to an oil. The latter was partitioned be-tween ethyl
acetate and water and the organic phase separated,
washed with sodium bicarbonate, brine, dried (MgS04)
and evaporated to an oil. Chromatography on silica
gel H (type 60) using a gradient of chloroform -to 4~0
methanol-chloroform afforded methyl 10,11 deoxypseudo-
monate as an oil (0.129 g), tlc in chloroform-methanol
9 : 1 showed one component at Rf = 0.46 and a single
Y P ~ max (C~IC13) 3400, 2900, 2850, 1720,
1~10, 1650, 1150 and 980 cm , Amax (EtOH) 221 nm
(E 11,500), ~H(CDC13) 5.75 (IH, m, C2-H), 5.40 (2H,
m, C10-H and Cll-H), 4.05 (2H, t, C97-CH2) 3.65 (3H,
s, CH30), 2.21 (3H, broad s, C15-CH3), 1.21 (3H, d,
C17-CH3) and 1.00 (3H, d, C14-CH3), ~c(CDCl3, 174.3
(Cl'), 166.8(Cl), 156.8(C3), 134.5 and 129.4 (clO and
11), 1~.7.6 (C2), 74.8 (C5), 71.2 (C13), 70.4 (C7),
2S 68.9 (C6), 64~8 (C16), 63.8 (C9'), 51.4 (CH30), 44.7
and 43.1 ~C4 and 12), 42.0 (C8), 34.1 (C27), 32.4
(C9), 29.1 (C41,5~ and 6'), 28.7 (C87), 26.0 (C7'),
24.9 (C3'), 20.4 'C14) 9 19.1 (C15) and 16.6 (C17),
m/e (relative intensity) 499 (100%, M+~l by C~
:
,:
Example 2
Pseudomonic acid_C b~ fexmentation
(a) fermen-tation
Pseudomon _ fluorescens, strain NCIB lOS86 was
cultured on an agar slope and flooded with sterile
water. A sample was added to the following seed staye
medium:
Oxoid*yeast extract 2% (w/v)
Glucose O.ll
lO Disodium hydrogen orthophosphate 0.2~
Potassium dyhydrogen orthophosphate 0.24
This was grown at 28C overnight and then used to inoc-
ulate the :~ollowing production stage medium:
Corn steep liquor 0.3~o (w/v)
15 Glucose 2.0
Glycerol O.S
Am~nonium sulphate 0.2
Calcium carbonate 0.4
Potassium dyhydrogen orthophosphate 0.04
20 Disodium hydrogen orthophosphate 0.0~5
Manganese chloride R H20 0.0003
Potassium chloride 0.05
Magnesium sulphate 7 H20 0.0375
P2000 to minimise foaming.
The fermen-tation was carried out at 25C for
48 hours when production was essentially complete.
Aftèr removing the cells by centrifugation the super-
natant was partitioned into ethyl acetate at pH 3. The
ethyl acetate solution was dried ~MgSO~) evaporated to
low volume, ether added and pseudomonic acid A allowed
to crystallise.
(b) Isolation of Pseudomonlc_acid C
The mother liquors from the above crystallization
* Trade Ma~k
~ ~ '
:
.; : . : . -
.
_ 15 -
were evaporated to an oil and chromatographed on
silica gel preparative 20 x 20 cm thin layer chromato-
graphy plates developed with chloroform : isopropanol :
acetic acid (80 : 20 : 0.5). The band above pseudomonic
acid A of Rf 0.65 was removed and re-chromatographed
as before to give pseudomonic acid C. [~ound: C, 6~.0;
H, 9.3%, C26H4408 requires C, 64.4; H9 9.2%], ~max
(CHC13) 3430 (broad, 1710, 1650, 1220 (broad), 1153,
1110, 1050, and 977 cm 1, ~max (EtOH) 222 nm ( 14,100)
~H(CDC13) 5.69 (lH, m, C2-H), 5.4 (2H, m, C10-H),
4.65 (4H, broad), 4.01 (2H, t, C9'-CH2), 2.15 (3H, S5
C15-CH3), 1-12 (3H, d, C17-CH3; J = 6 Hz), 0.96 (3H,
d, C14-CH3; J = 8 Hz), ~C (CDC13) 178.1 (Cl~), 166.9
(Cl), 156.9 (C3), 134.5 and 129.5 (C10 and Cll), 117.6
(C2), 74.9 (C5), 71.4 (C13), 70.4 (C7), 69.0 (C6), 64.9
~C16), 63~9 (C9~), 44.7 (C12), 43.0 (C~), 41.9 (C8),
34.0 (C2'), 32.4 (C9), 28.9 (C4', 5' and 6'), 28.6
(C8~), 25.9 (C7'), 24.7 (C3'), 20.4 (C14), 19.2 (C15),
16.7 (C17).
Examele 3
Isolation o~ ~S~ Y~b~Y~ LY~a
The residual oil from the mother liquors from
Example 2(a) (ca 5 y) was dissolved in methanol (50 ml),
diluted with water (50 ml) and the pH adjusted to 7
with aqueous sodium hydroxide. After evaporation to
dryness a solution of the residue in dimethylformamide
(50 ml), hexame-thylphosphoramide (5 drops) and methyl
iodide (5 ml) was stirred overnight at room temperature.
The solution was evaporated to dryness and the residue
partitioned between ethyl acetate (50 ml) and water
(20 ml). The organic layer was washed with saturated
brine, aqueous sodium bicarbonate, brine, dried (MyS04)~
and evaporated to an oil from which some methyl
.
.
'
.. .
- : .... ' . ' '
'7
- 16 -
pseudomonic A crystallised. The residual oil was
chromatographed twice on silica (20 g then 15 g,
type 60) eluting with gradient of O - ~% methanol -
chloroform. ~ractions con-taining substantially pure
methyl pseudomonate C (Rf 0.4~ silica tlc, cnloroform/
methanol 9 : 1, methylpseudomonate A Rf 0.42) were com-
bined and chromatographed on silica (~ g, type 60) using
gradient of O - 3% methanol - chloroform (distilled
from phosphorus pentoxide). Fractions containing pure
methyl pseudomonate C (by tlc) were combined and evap-
orated to an oil (50 mgs) which was found to be spectro-
scropically and chromatographically identical to methyl
10~11-deoxypseudomonate A obtained -in Example 1.
Example 4
Pseudomonic Acid C from meth~1 pseudomonate_C
OH
HO~ ~ O ~ ~ OH
~ ' O
OH
Methyl pseudomonate C (230 mgs) from Example 3
was dissolved in DM~ (25 ml) and diluted with 0.05 M
phosphate buffer (120 ml) then stirred with Bakers ~east
(6 g) overnight. The mixture was filtered, evaporated
to dryness and the residue dissolved in ethyl acetate
(50 ml) and water (50 ml). The organic layer was washed
with water and combined aqueous layers adjusted to pH
3 (5M HC1) and extxacted several times with e-thyl
acetate. After drying the combined extracts were
evaporated to yield an oil. Chromatography on silica
. ' .
:: : .: . ' " : ' ~. ~ :
_ 17 -
gel H (type 60, 8 g) eluting with gradient 0 -to 6%
methanol - chloroform gave pseudomonic acid C (150 mgs,
67%) which was chromatographically and spectroscopically
identical to ma-terial obtained in Example 2.
~ æ~
Methyl Pseudomonate C
(Alternative procedure to Example 3)
Mother liquors (15 g) from Example 2 (a) were
dissolved in acetone (150 ml), and stirred overnight
with potassium carbonate (42 g) and methyl iodide (21 ml)
at room temperature. The reaction rnixture was f;.ltered
and the iltrate evaporated to dryness then taken up i.n
ethyl acetate/water and worked up as in Exa~ple 3 to
give methyl pseudomonate C.
The methyl ester may be hydrolysed as in Example 4.
Sodium Pseudomonate C
_
(Sodium 10,11-deoxypseudomonate A)
A soluti.on of methyl pseudomonate C (0.330 g) in
distilled tetra-hydrofuran (20 ml) and N/10 sodium
hydroxide solutlon (20 ml) was stirred at room temper-
ature for 2 hours. The tetrahydrofuran was removed ',
in vacuo to give a turbid aqueous solution, which was
washed with,ether-ethyl acetate to remove unreacted
ester~ The aqueous layer was saturated with sodium
chloride~ layered wlth ethyl acetate and-acidified with
dilute hydrochloric acid to pH 1.5. The ethyl acetate
layer was separated, washed with brine, dried (MgS04)
., .
.
- -' ' ' ' ' : ~
.
'
'7
- 18 -
and evaporated to dryness. The pseudomonic acid C
obtained was suspended in water - tetrahydrofuran and
N/10 sodium hydroxide solution added to pH 7.5. The
resulting aqueous solution was evaporated to dryness
in vacuo. The residue was dissolved in dry methanol
(5 ml) filtered and excess dry ether added to the
filtrate with stirring. ~he resulting white precipi-
tate of sodium pseudomonate C was collected and dried
in vacuo (0.153 g). The compound was homogeneous by
thin layer chromatography and high pressure liquid
chromatography, [~]D ~ 0.94 (c 1.0, MeOH), vmax (KBr)
3400, 2800, 1700, 1640, 1560, 1230, 1150, 975 cm~1,
x (BtOH) 221 nm (~ 13,300)~ ~H((CD3)2SO)) 5-7
(lH, m, vinylic-~), 5.3 (2H, m, -CH=CH-), 3.95 (2H,
5, CH2-9~), 2.08 (3H, s, vinylic -CH3), 0.95 ~3H, d,
secondary -CH3) and 0.88 (3H, d, secondary -CH3).
Example 7
Sodium P_eudomonate`~C
Methyl pseudomonate C (1 g) was dissolved in TH~
(50 ml)/water (50 ml) and the pH adjusted to 12 and
maintained for 21~ hours. The solution was adjusted to
pH7 and evaporated to dryness then redissolved in
water (30 ml) and washed with ethylacetate. The aqueous
fraction was then acidified to pH2 and extracted with
ethyl acetate. After drying (MgS04) the combined
extracts were evaporated i.n vacuo. The resultant oil
(0.55 g) was treated with sodium bicarbonate (95 mgs,
1 eq) in water (20 ml)/methanol (20 ml) and evaporated
to dryness. The sodium salt was dissolved in ethanol
(minimum~ and added dropwise to ether ~300 ml) and the
precipitate filtered off (0.58 g, 57%) ? vmax (KBr)
3380 (broad), 1700, 1642, 1560, 1225~ 1150 and 973 cm
... .- - ~.. . . .
'7
-- 19 --
~max (EtOH) 222 nm (~ 13,700), ~H(CH30D) 1.07 (3H, d9
J 7Hz~ C17-CH3), 1-18 (3H, d, J 7H~, C14-CH3), 1.44
(12H, m,-~CH2)6), 2.25 (3H, s, C15-CH3), 4-11 (2H~ 5,
C9-C~I~), 5.5 (2H, m, }1-10, H-ll), 5.79 (lH, s, H-2),
~c (CD30D) 183.0 (Cl7), i68.4 (~1), 158.9 (C3), 135.7,
129.6 (C10, Cll), 118,2 (C2), 76.2 (C5)9 72.0 (C7), 71.
(C13), 69.8 (C6), 65.6 (C16), 64.8 (C9l), 45.2 (C12),
44.0, 43.6 (C4, C8), 39.3 (C2~), 33.6 (C9), 30.7, 30.4,
30.3, 29.8, 27.7, 27.1 (C37-C87), 20.3 (C14). 19.4 ~C15),
16.6 (C17) (~ound: C, 59.4; H, 8.4; Na, 4.9~C26H4308
Na.H20 requires C, 59.5; H, 8.6; Na, 4.4%).
Example 8
Pseudomonic acld C
Pseudomonic acid A (500 mgs) was dissolved in
2,2-dimethoxypropane (20 ml) and ethyl acetate (20 ml)
then ~-toluene sulphonic acid (few crystals) added.
The solution was stirred for 1 hour then washed with
brine and dried (MgS04). After evaporation of the sol-
vent in vacuo, the residue was dissolved in water-
methanol (1:1, 20 ml) and potassium bicarbonate (100 mgs,
1 eq) added. The solvents were removed in v~cuo and
potassium selenocyanate (432 mgs, 3 eq), tert-amyl al-
cohol-water (9:1, 15 ml) added and the reaction
~ refluxed for 4 days. Af~er filtering the solution
was evaporated to dryness, water (20 ml) added and
solution adjusted to pH2 (5M-HCl) under a layer of
ethyl acetate (20 ml). The organic layer was separated
and the aqueous layer further extracted with ethyl
acetate (3 x 20 ml). The combined extracts were dried
(MgS04) and evaporated to an oil which was redissolved
in ~0% acetic acid (10 ml) and stirred at room temp~
exature overnight. The solution was evaporated to
dryness and the residual oil chromatographed on silica
-:: , ~ , ,, - ' : ' ' : :
. , . ~. ,
. .
:
~ t7
- 20 -
(10 g) eluting with 0 to 6% methanol-chloroform.
~ractions containing pure product (tlc) were combined
and evaporated to give pseudomonic acid C (280 mgs,
58%).
Example 9
Pseudomonic acid C
Pseudomonic acid A (500 mgs) was dissolved in
2,2-dimethyoxypropane (50 ml) and treated with p-toluene
sulphonic acid (few crystals). After 1 hour the
solution was diluted with ethyl acetate, washed with
brine and dried (MgS0~). The solution was evaporated
in vacuo and the residue redissolved in water-methanol
(1:1, 20 ml) and potassium bicarbonate (100 mgs, 1 eq)
added. The solvents were removed in vacuo and potas-
sium selenocyanate (432 mgs 7 3 eq) and iso-hexylalcohol-
water (9:1, 15 ml) added and reaction refluxed for 4
days. After filtering, the reaction mixture was diluted
with ethyl acetate (20 ml~ and extracted with aqueous
sodium bicarbonate (3 x 20 ml). The aqueous extracts
were combined and acidified with acetic acid under a
layer o~ ethylacetate (20 ml). After stirring for ca.
1 hour the organic layer was separated and aqueous
layer further extracted with ethylacetate (3 x 20 ml).
The combined extracts were washed with hrine, dried
(MgS04) and evaporated to an oil`. Chromatography of
the oil on silica (5 g) gave pure pseudomonic acid C
(215 mgs, 45%).
.: ~ , . . :
,~ . ' ... ' . : ,
.
,, . . . .: .
Example 10
Methyl pseudomonate C
Methyl pseudomonate A (5.14 g), potassium selen-
ocyanate (4.32 9) in iso~hexyl alcohol - water (9:1,
150 ml) were refluxed for 3 days. The reaction mixture
was filtered then evaporated to dryness and dissolved
in ethyl acetate (50 ml) - brine (50 ml)0 The organic
layer was separated, washed with brine (50 ml) then
dried (MgS04). After evaporation of the solvents 1n
vacuo, the xesidue was chromatographed on silica (80 g)
eluting with 0 - 4% methanol-chloroform. Pure fractions
were combined and evaporated to an oil which on stand-
ing gave crystalline r~ethyl pseudomonate C, mp. ~L7-9C
(2.57 g, 52%) (~ound: C, 65.0; H, 9.5. C27H4608 req-
uires C, 65.0; ~I, 9.3%).
Exam~le 11
Methyl_pseudomonate C and pseudomonic acid ~
Methyl pseudomonate A (514 mgs) was dissolved in
2,2-dimethoxypropane (20 ml) and a few crystals of p~
toluene sulphonic acid added. The solution was stirred
for 1~ hour then ethyl acetate (20 ml) added and -the
solution washed with brine then dried (MgS04). After
evaporation of the solvents~ the acetonide was dissolved
in tert-amyl alcohol-water (9:1, 15 ml), potasslum
selenocyanate (432 mgs) added and reaction refluxed ~or
5 days. The solution was filtered, evaporated to
dryness and the residue dissolved in ethyl acetate
(20 ml)-brine (20 ml). The organic layer was separated
and the aqueous layer further extracted with ethyl
acetate (3 x 20 ml). The combined extracts were dried
(MgS04) and evaporated to dryness. The crude product
. ' . : .' ~ , -
,. ' - '
.~
. ~ . . .
:
~ 5
- 22 -
contained two major components (tlc) which were sep-
arated by column chromatography o~ silica (10 g) elut-
ing ~ith O - 8% methanol-chloro~orm. The first fraction
was identified as methyl 6,7-0-isopropylidene pseu~o-
~Gnate C (206 mgs, 38%), ~max (CHC:L3) 34503 1722,
1643 and 1220 cm 1, ~H~CDC13) 0.98 (3H, d, J 7Hz, C17-
CH3), 1.13 (3H, d, J 7Hz, C14-CH3), 1.33 (15H, m,
(CH2)6, acetonide CH3), 1.48 (3H, s, ace-tonide C_3),
2.18 (3H, s, C15-CH3), 3 63 (3H, s, OCH3), 4.05 (2H,
19 t, C91-CH2), 5.45 (2H, m, H-10, H-ll), 5.73 (lH9 s,
H-2), ~c(CDCl3) 174.0 (Cl'), 166.6 (Cl), 156.2 (C3),
135.G9 128.9 (C10, Cll), 117.8 (C2), 108.7 (~ C ~ ),
76.5 (C5), 76.7 (C7), 74.3 (C6), 71.0 (C13), 66.5
(C16)~ 63-7 (C9~)s 51-3 (OCH3), 44.6 (C12), 44.1 (C4),
37.9 (C8), 34.2 (C2 9 ) ~ 34.1 (C9), 29-1 (C41~ 51~ 61)9
28.8 (C8l), 28.3, 26.3 (,,C ~MMee), 26.0 (C7'), 24.9 (C3')9
20.3 (C14), 19.1 (C15), 16.4 (Cl7), ~ e (relative int-
ensity) 523 (6%), 494 (19), 436 (22), 369 (30), 306
~22), 299 (20) (~ound: 523.3263~ M~-CH3 requires
523.3257), The second fraction was identified as
6,7-0-isopropylidene pseudomonic acid C (130 mgs,
2S%), ~ ax (CHC13) 2300-3600 (broad), 1702, 1642,
1220, 1152 and 1052 cm 1, ~H (CDC13) 0.98 (3H, d, J
7Hz, C17-CH3), 1.14 (3H, d, J 7Hz, C14-CH3), 1.33
(15H, m, ~CH2)6, acetonide CH3), 1.49 (3H, S9 acetonide
CH3), 2.18 (3H, s, C15-CH3), 4-06 (2H~ t, C91-CH2)'
5.45 (2H, m, H-10, H-ll), 5.73 (lH, s, H-2), ~c (CDC13)
178.1 (Cl'), 166.7 (C~), 156.1 (C3)9 134.g, 129.0
(C10, Cll), 117.8 (C2), 108.7 (= C ~), 76.4 (C5), 75.6
(C7), 74.2 (C6), 71.2 (C13),66.4 (C16), 63.8 (C9~),
44.4 (C12), 44.0 (C4), 36-8 (C8), 34.0 ~C21), 33.7 (C9),
29.0 (C41~ 51, 6l), 28-7 (C81), 28.3, 26.2 (_~C--M )'
25.9 ~C7l), 24.7 (C3'), 20.1 (Cl~), 19.0 (C15), 16.4
(C17~, ~ e (relative intensity) 509 (7%), 480 (10),
422 (10), 404 (8), 394 (10), 387 (7~, 383 (8). (~ound:
509.3110. M+-CH3 requires 509.3108~. The acetonides
, -
'
_ 23 -
were quantitatively converted to methyl pseudomonate C
and pseudomonic acid C respectively with 80% acetic
acid overnight.
Example 12
Methyl pseudomo a_e C
Sodium iodide (600 mgs, 4 eq) (dried at 110C/4
hours) was stirred in dry THF (1 ml) and dry acetoni-
trile (1 ml) and trifluoroacetic anhydride (141 ~1,
1 eq) was added. After 5 minutes the yellow solution
was cooled in ice and methyl pseudomonate A (514 mgs)
added. After 5 minutes the ice bath was removed and
the reaction stirred at room temperature for 24 hours.
The reaction mixture was diluted with aqueous sodium
bisulph:ite arld extracted with ethyl ace~ate (4 x 25 ml).
The combined extracts were washed with brine then dried
(MgS04) and evaporated to an oil then chromatographed
on silica (5 g). Pure fractions (tlc, hplc) ~ere com-
bined and evaporated to give desired product (63 mgs,
13%).
Isohexy~ pseudomonate_C
Methyl pseudomonate A (10 g) and po-tassium sel-
enocyanate (4.32 g~ 1.5 eq) in 2-ethyl-n-butanol (iso-
hexyl alcohol)-water (9:1, 150 ml) were refluxed for
2 days. The solution was filtered and evaporated then
the residue redissolved in ethyl acetate~water. The
organic layer was separated, washed with brine then
dried (MgS04) and the solvent removed in vacuo. The
crude product was chroFatographed on silica (100 g)
. . :
- 24 _
elutin~ with 0 to 6% MeOH-CHC13. Fractions containing
pure methyl pseudomonate C were combined and evaporated
to an oil which crystallised on standing mp 47-9C
(2.2 g). Remaining fractions were combined and rechrom-
atographed to yield a pure compound subsequently iden-
tified as isohexyl pseudomonate C (1.0 g) vmax (CHC13)
3400 (broad), 1703, 1640, 1427, 1220, 1150, 1020 and
978 cm 1, ~H (CDC13) 0.94 (6H, t, (CH2CH3)2), 0-97
(3H, d, C17-CH3), 1.14 (3H, d, C14-CH3), 1~32 (12H~
m.-(CH2)6-), 2-19 (3H, s, C15-C_3), 3.95 (2H, d,
OCH2CHEt2) 9 4.05 (2H, t, C9~-CH2), 5-45 (2H~ m~ H-10~
H-ll) 5-75 (lH, S9 H-2), ~c (CDC13) 174.0 (C1~3, 166.9
(C13, 157.2 (C3), 134.4, 129.0 (C10, Cll), 117.8 (C2),
75~0 (C5), 71.2 (C13), 70.4 (C7), 68.8 (C6), 6603
(0_~12CHEt2), 64.9 (C16), 63.8 (C9'), 44.4 (C12), 43.2
(C4), 42-1 (C8), 40.5 (OCH2C~IEt2), 34.4 (C2'), 32.5
(C9), 29.1 (C4', C597 C6l), 28.8 (C8'), 26.0 (C7l),
25-0 (C3~), 23-4 (CH2CH3) 20.3 (C14), 19.2 (C15),
16.4 (C17), 11.0 (CH2CH3).
:. . . ., :. .. . ..
.:
: . : .
, ,. .,, - : ., . . . : . -
- 25 -
BIOLOGICAL DATA
(1) Antibacterial activity - human or~anisms
Table 1 shows the antibacterial spectrum of
sodium pseudomonate C and methyl pseudomonate C in
terms of minimum inhibitory concentration (~g/ml)
measured by serial dilution in nutrient agar con-
taining 5% chocolated horse blood.
TABLE 1
_ _ _ . ___
ORGANISM M.I~C. (~g/ml)
__ _ ._
sodium methyl
pseudo- pseudo-
monate C monate C
E. coli NCTC 10418 >100 >100
E. coli ESS 1.0 2.5
P. mirabilis 889 >100 >100
K. aerogenes A >100 >100
Ps aeruginosa NCTC 10701>100 ~100
Pasteurella multocida 1633 1.0 2.5
Haemophilus influenzae Ql0.1 <0.2
Haeomophilus influenzae Wy 21 0.1 0.5
Neisseria flavescens 66330.2 0.5
Bacillus subtilis 0.02 <0.2
Corynebacterium xerosis 9755 ~100 ~100
Sarcina lutea ~100 ~100
Staph. aureus Oxford 0.1 <0.2
Staph. aureus Russell 0.2 0.5
Staph. aureus 1517 0.2 0.5
Strep. faecalis I 100 ~100
Strep. Pyogenes A 64/848 0.1 1.0
Strep. Pyogenes B 2788 2.5 1.0
Strep. Pyogenes C 2761 0.2 1.0
Strep. pneumoniae CN33 0.1 Ool
__ __ _ _ .... . ~_ _
., ~' '
,
Y~ 7
- 26 -
(2) Anti-myco~asma activity
Methyl pseudomonate C possess good antimyco
plasma activity in vitro against mycoplasmas ~om
human and veterinary sources, as shown in table 2.
Method
(1) The minimum inhibitory concentrations ~MIC) of
methyl pseudomonate C were determined in Microtitre
plates, by a modification of the metabolic-inhibition
test (Taylor-Robinson, 1967). The compounds were
serially diluted in sterile de-ionised water to give a
range of concentrations from 250-0.5 ~g/ml. Mycoplasma
broth containing 1% (w/v~ of glucose and 0.005% (w/v)
of phenon red, was added at a strength to compensate
for its dilution by the aqueous drug solution. Appro~-
imately 10~ colony forming units of mycoplasma were
added to each concentration of dxug. Drug-free infected,
non-infec-ted and pH control wells were included on each
plate. Plates were sealed with cellophane tape and
incubated at 37C for seven days. The MIC was the
lowest concentration of compound that prevented a colour
change in the mycoplasma broth, caused by the metabolism.
Reference
~_ .
Taylor-Robinson, 1967. Mycoplasmas of various hosts
and their antibiotic sensitivities. Post. Grad. Med. J.,
43 Suppl. CMarch~, 100.
. : , , ' '.,, :. .: .' ' ` .
' , ' ' .'., :.: . . , ~: ..
' '' ' ' ' . '....... ' :' -' - .'' ' ' . '' ' '
.. : ., . . :. , . - . .... . .
_ 27 -
TABLE 2
. _ ~_
MYCOPLASMA M.I.C. (~g/ml)
__ _ __
M. gallisepticum S.6 62.5
M. synoviae 25204 ~0.5
M. pulmonis JB <0.5
M. suipneumoni~e L~ber <0.5
M. pneumoniae 427a 1.0
M. fermentans MW KL4 <0.5
Table 3 shows MIC values for sodium pseudomonate
C and met'nyl pseudomonate C against further mycoplasma
species determined in Friis~ broth using the micro-
titer method.
TABLE 3
ORGANISM M.I.C (~g/ml)
. ~ , .
MethylSodium
Pseudo-Pseudo-
monate Cmonate C
M. suipneumoniae Str. 11 ~10 ~10
M. suipneumoniae J2206/183b >10 10
M. dispar H225 10 5.0
M. dispar NCTC 10125 50 2.5
M. pneumoniae 427a ~10 2.5
M. pneumoniae ATCC 15492 10 -
M. fermentans MWKL4 0.039< 0.02
M. pulmonis JB 0.3120,039
. ~
.
.' ~ ,' . : ' .. " :, ' ~, ...... ... .
.. . . . . .
